Immunocytochemical study of Ki-67 as a prognostic marker in canine mammary neoplasia

Background: Canine mammary tumors are challenging for clinicians and pathologists because of complex histologic classification, low specificity of cytologic diagnosis, and unpredictable biological behavior. In histologic specimens, expression of tumor proliferation marker Ki-67, a nuclear nonhistone protein, has been shown to have prognostic value for canine mammary tumors and to correlate with malignancy and low survival rates. Objective: The objective of this study was to measure the proliferation index of canine mammary tumors by immunochemical detection of Ki-67 in cytologic specimens and to determine its relationship to clinical and pathologic variables and patient outcome. Methods: Spontaneous mammary tumors from 31 female dogs were surgically excised. Imprint specimens for cytologic evaluation were wet-fixed in ethanol; histologic specimens were prepared routinely. Immunostaining was performed with the PH 177 monoclonal antibody against Ki-67; proliferation index was graded from negative to +++. Dogs were followed for 18 months. Multivariate logistic regression analysis was used to determine correlations between immunocytochemical results, tumor and clinical variables, and patient outcome. Results: Ki-67 proliferation indices in cytologic specimens were significantly lower for nonmalignant tumors than for malignant tumors. High index values of Ki-67 were positively correlated with metastasis, death from neoplasia, low disease-free survival rates, and low overall survival rate. With the exception of 4 specimens for which cellularity was insufficient, positive expression of Ki-67 in cytologic specimens correlated with that of histologic specimens. Conclusions: The prognostic value of the Ki-67 index in canine mammary tumors by using wet-fixed cytology imprint specimens was similar to that observed previously for histologic specimens. Immunocytochemical detection of Ki-67 could improve the accuracy and value of cytology by providing safe and rapid information about malignancy and patient outcome. © 2004 American Society for Veterinary Clinical Pathology

The role of melatonin on miRNAs modulation in triple-negative breast cancer cells.

Melatonin, a hormone secreted by pineal gland, exerts antimetastatic effects by reducing tumor cell proliferation, migration and invasion. MicroRNAs (miRNAs) are small, non-coding RNAs that play a crucial role in regulation of gene expression and biological processes of the cells. Herein, we search for a link between the tumor/metastatic-suppressive actions of melatonin and miRNA expression in triple-negative breast cancer cells. We demonstrated that melatonin exerts its anti-tumor actions by reducing proliferation, migration and c-Myc expression of triple negative breast cancer cells. By using Taqman-based assays, we analyzed the expression levels of a set of miRNAs following melatonin treatment of triple negative breast cancer cells and we identified 17 differentially expressed miRNAs, 6 down-regulated and 11 up-regulated. We focused on the anti-metastatic miR-148b and the oncogenic miR-210 both up-regulated by melatonin treatment and studied the effect of their modulation on melatonin-mediated impairment of tumor progression. Surprisingly, when miR-148b or miR-210 were depleted in triple-negative breast cancer cells, using a specific miR-148b sponge or anti-miR-210, melatonin effects on migration inhibition and c-myc downregulation were still visible suggesting that the increase of miR-148b and miR-210 expression observed following melatonin treatment was not required for the efficacy of melatonin action. Nevertheless, ours results suggest that melatonin exhibit a compound for metastatic trait inhibition, especially in MDA-MB-231 breast cancer cells even if a direct link between modulation of expression of certain proteins or miRNAs and melatonin effects has still to be established

Expression of glutathione, glutathione peroxidase and glutathione S-transferase pi in canine mammary tumors

Background: Glutathione (GSH) is one of the most important agents of the antioxidant defense system of the cell because, in conjunction with the enzymes glutathione peroxidase (GSH-Px) and glutathione S transferase pi (GSTpi), it plays a central role in the detoxification and biotransformation of chemotherapeutic drugs. This study evaluated the expression of GSH and the GSH-Px and GSTpi enzymes by immunohistochemistry in 30 canine mammary tumors, relating the clinicopathological parameters, clinical outcome and survival of the bitches. In an in vitro study, the expression of the genes glutamate cysteine ligase (GCLC) and glutathione synthetase (GSS) that synthesize GSH and GSH-Px gene were verified by qPCR and subjected to treatment with doxorubicin, to check the resistance of cancer cells to chemotherapy.Results: The immunohistochemical expression of GSH, GSH-Px and GSTpi was compared with the clinical and pathological characteristics and the clinical outcome in the bitches, including metastasis and death. The results showed that high immunoexpression of GSH was correlated to the absence of tumor ulceration and was present in dogs without metastasis (P < 0.05). There was significant correlation of survival with the increase of GSH (P < 0.05). The expression of the GSH-Px and GSTpi enzymes showed no statistically significant correlation with the analyzed variables (p > 0.05). The analysis of the relative expression of genes responsible for the synthesis of GSH (GCLC and GSS) and GSH-Px by quantitative PCR was done with cultured cells of 10 tumor fragments from dogs with mammary tumors.The culture cells showed a decrease in GCLC and GSS expression when compared with no treated cells (P < 0.05). High GSH immunoexpression was associated with better clinical outcomes.Conclusion: Therefore, high expression of the GSH seems to play an important role in the clinical outcome of patients with mammary tumors and suggest its use as prognostic marker. The in vitro doxorubicin treatment significantly reduces the expression of GCLC and GSS genes so we can consider them to be candidates for predictive markers of therapeutic response in mammary cancer

HET0016, a Selective Inhibitor of 20-HETE Synthesis, Decreases Pro-Angiogenic Factors and Inhibits Growth of Triple Negative Breast Cancer in Mice

<div><p>A selective inhibitor of 20-HETE synthesis, HET0016, has been reported to inhibit angiogenesis. 20-HETE has been known as a second mitogenic messenger of angiogenesis inducing growth factors. HET0016 effects were analyzed on MDA-MB-231 derived breast cancer in mouse and <i>in</i><i>vitro</i> cell line. MDA-MB-231 tumor cells were implanted in animals’ right flank and randomly assigned to early (1 and 2), starting treatments on day 0, or delayed groups (3 and 4) on day 8 after implantation of tumor. Animals received HET0016 (10 mg/kg) treatment via intraperitoneal injection for 5 days/week for either 3 or 4 weeks. Control group received vehicle treatment. Tumor sizes were measured on days 7, 14, 21, and 28 and the animals were euthanized on day 22 and 29. Proteins were extracted from the whole tumor and from cells treated with 10 µM HET0016 for 4 and 24 hrs. Protein array kits of 20 different cytokines/factors were used. ELISA was performed to observe the HIF-1α and MMP-2 protein expression. Other markers were confirmed by IHC. HET0016 significantly inhibited tumor growth in all treatment groups at all-time points compared to control (p<0.05). Tumor growth was completely inhibited on three of ten animals on early treatment group. Treatment groups showed significantly lower expression of pro-angiogenic factors compared to control at 21 days; however, there was no significant difference in HIF-1α expression after treatments. Similar results were found <i>in</i><i>vitro</i> at 24 hrs of HET0016 treatment. After 28 days, significant increase of angiogenin, angiopoietin-1/2, EGF-R and IGF-1 pro-angiogenic factors were found (p<0.05) compared to control, as well as an higher intensity of all factors were found when compared to that of 21 day’s data, suggesting a treatment resistance. HET0016 inhibited tumor growth by reducing expression of different set of pro-angiogenic factors; however, a resistance to treatment seemed to happen after 21 days.</p></div

Immunohistochemistry stained with Ki-67 in vehicle and HET0016 treated tumors groups.

<p><b>A)</b> Quantitative estimation of cell proliferation showing number of cells per area (m²). <b>B)</b> Top images were taken from tumor of 21 days and bottom images were taken from tumor of 28 days. The arrows shows cellular nucleus stained. Error bars: ± standard error, **p<0.001. Scale bar = 100 µm.</p

Immunohistochemistry stained with vWF in vehicle and HET0016 treated tumors groups.

<p><b>A)</b> Quantitative estimation of Mean Vascular Density. <b>B)</b> Top images were taken from tumor of 21 days and bottom images were taken from tumor of 28 days. The arrows shows micro-vessels. Error bars: ± standard error, **p<0.001. Scale bar = 100 µm.</p

Human cytokine antibody array kit (RayBiotech, USA) contending 20 different growth factors/cytokines were used for analysis of protein expression profile in tumor tissue and cells lysate.

<p>G-CSF: colony stimulating factor; PDGF-RA: platelet-derived growth factor receptor, alpha polypeptide; PDGF-AA: platelet-derived growth factor alpha polypeptide; bFGF: fibroblast growth factor (basic); EGF: epidermal growth factor; EGF-R: epidermal growth factor receptor; IGF-I: insulin-like growth factor 1; MMP-9: matrix metallopeptidase 9; SDF-1α: Stromal cell-derived factor 1 alpha; Tie-1: receptor tyrosine kinase of angiopoietin-2; Tie-2: receptor tyrosine kinase of angiopoietin-1; VEGF-A: vascular endothelial growth factor A; VEGF-C: vascular endothelial growth factor C; VEGF-R2: vascular endothelial growth factor receptor 2; VEGF-R3: vascular endothelial growth factor receptor 3. All antibodies are prepared in duplicate. ns: no significant; *not evaluated.</p><p>Human cytokine antibody array kit (RayBiotech, USA) contending 20 different growth factors/cytokines were used for analysis of protein expression profile in tumor tissue and cells lysate.</p

Determination of (A) HIF-1 and (B) MMP-2.

<p>No significant differences were observed among the treatment groups.</p

HET0016 treatment reduced tumor growth in breast cancer nude mice.

<p><b>A)</b> HET0016 treatment decreased tumor volume in both the early and delayed treatment groups. Significant difference was observed between control and treatment groups as early as 7 days of treatment. When compared the tumor growth rates (<b>B</b>), animal that received delayed treatment showed rapid growth at late stage of treatment. <b>C)</b> Representative samples of mammary tumors developed by MDA-MB-231 cells implantation on the right flank of mice. **p<0.001, ***p<0.0001.</p